Biomedical subjects
Publications and source records attributed to A M Krensky.
In situ hybridization was used to localize the lymphocyte activation gene 519 (D2S69E) on human metaphase chromosomes. A significant proportion of the grains were situated over chromosome 2p12----q11, a region which contains other genes involved in immunologic functions.
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CTL are thought to play a role in the elimination of transformed cells in vivo. The effectiveness of such CTL is in part dependent on recognition of tumor specific antigens. Among the best characterized tumor-specific antigens are the unique or idiotypic determinants on the Ig of B cell lymphomas. Here we describe the generation and properties of human CTL specific for the idiotype on autologous B cell tumors. These cells are CD3+,CD4-,CD8- and express the delta chain of the TCR. Such cells may prove useful in tumor-specific adoptive therapy.
Cytotoxic T lymphocytes (CTL) expressing the CD8 glycoprotein recognize peptide antigens presented by class I major histocompatibility complex (MHC) molecules. This correlation and the absence of CD8 polymorphism led to the hypothesis that CD8 binds to a conserved site of class I MHC molecules. Using a cell-cell binding assay we previously demonstrated specific interaction between human class I MHC (HLA-A,B,C) molecules and CD8. Subsequent analysis of the products of 17 HLA-A,B alleles revealed a natural polymorphism for CD8 binding in the human population. Two molecules, HLA-Aw68.1 and HLA-Aw68.2, which do not bind CD8, have a valine residue at position 245 whereas all other HLA-A,B,C molecules have alanine. Site-directed mutagenesis shows that this single substitution in the alpha 3 domain is responsible for the CD8 binding phenotype and also affects recognition by alloreactive and influenza-specific CTL. Our results indicate that CD8 binds to the alpha 3 domain of class I MHC molecules.
HLA-A2.1 and HLA-A2.3, which differ from one another at residues 149, 152, and 156, can be distinguished by the mAb CR11-351 and many allogeneic and xenogeneic CTL. Site-directed mutagenesis was used to incorporate several different amino acid substitutions at each of these positions in HLA-A2.1 to evaluate their relative importance to serologic and CTL-defined epitopes. Recognition by mAb CR11-351 was completely lost when Thr but not Pro was substituted for Ala149. A model to explain this result based on the 3-dimensional structure of HLA-A2.1 is presented. In screening eight other mAb, only the substitutions of Pro for Val152 or Gly for Leu156 led to the loss of mAb binding. Because other non-conservative substitutions at these same positions had no effect, these results suggest that the loss of serologic epitopes is in many cases due to a more indirect effect on molecular conformation. Specificity analysis using 28 HLA-A2.1-specific alloreactive and xenoreactive CTL clones showed 19 distinct patterns of recognition. The epitopes recognized by alloreactive CTL clones demonstrated a pronounced effect by all substitutions at residue 152, including the very conservation substitution of Ala for Val. Overall, the most disruptive substitution at amino acid residue 152 was Pro, followed by Glu, Gln, and then Ala. In contrast, substitutions at 156 had little or no effect on allogeneic CTL recognition, and most clones tolerated either Gly, Ser, or Trp at this position. Similar results were seen using a panel of murine HLA-A2.1-specific CTL clones, except that substitutions at position 156 had a greater effect. The most disruptive substitution was Trp, followed by Ser and then Gly. In addition, when assessed on the entire panel of CTL, the effects of Glu and Gln substitutions at position 152 demonstrated that the introduction of a charge difference is no more disruptive than a comparable change in side chain structure that does not alter charge. Taken together, these results indicate that the effect of amino acid replacements at positions 152 and 156 on CTL-defined epitopes depends strongly on the nature of the substitution. Thus, considerable caution must be exercised in evaluating the significance of particular positions on the basis of single mutations. Nonetheless, the more extensive analysis conducted here indicates that there are differences among residues in the class I Ag "binding pocket," with residue 152 playing a relatively more important role in formation of allogeneic CTL-defined epitopes than residue 156.
Lymphocyte function-associated antigen 1 (LFA-1) is a glycoprotein involved in virtually all aspects of the immune response requiring direct cell to cell contact. It has been suggested that lack of LFA-1 expression in lymphomas may represent a mechanism of escape from immunologic surveillance. We investigated the expression of LFA-1 in a series of more than 250 lymphoid neoplasms and reactive lymphoid proliferations using a frozen section immunoperoxidase technique. LFA-1 was expressed by all lymphoid populations in the reactive cases. In contrast, absence of LFA-1 alpha or beta chains was found in 44% of non-Hodgkin's lymphomas, including 50% of B-cell lymphomas. These findings suggest that loss of LFA-1 expression may be of great use in the differential diagnosis of benign versus malignant lymphoproliferations. Eighty percent of initial biopsy specimens of low-grade lymphoma exhibited LFA-1 expression, whereas only 8% of recurrent specimens retained expression of both LFA-1 subunits. However, we found no correlation between LFA-1 expression and clinical course in a series of 64 patients with diffuse large cell lymphomas.
Human cytolytic T lymphocytes (CTL) were generated in the presence and absence of histamine in order to define the role of this autacoid in immune regulation. Histamine (10(-8)-10(-4) M) suppressed the generation of class I specific CTL but, at 10(-4) M, actually increased class II specific cytolysis. Histamine acted at the level of CTL generation; histamine was not present in the cytolytic assay. When histamine was added to the cytolytic assay with CTL grown without histamine, the lytic ability of the effector cells was similar to that of controls. Histamine-induced suppression of class I specific cytolysis was blocked by continuous culture with the H2 antagonist ranitidine but not with the H1 antagonist pyrilamine. These data suggest that suppression was mediated by the H2 receptor. Continuous culture with histamine had no effect on T cell proliferation or the expression of cell surface molecules. Histamine-induced suppression of class I specific cytolysis was reversed by the addition of PHA to the cytotoxicity assay, showing that the cytolytic machinery was intact. These data provide evidence that histamine is involved in regulation of cytolytic T cells.
In normal kidneys, peritubular and glomerular capillaries can be readily identified by their intense expression of HLA class I and class II compared to other cells within the graft. This high density of expression of MHC, plus their exposure to activated circulating lymphocytes, makes these cells the likely early and primary target of rejection responses. The fate of these capillaries during renal allograft rejection was examined using an indirect immunoperoxidase staining technique and monoclonal antibodies to class I and class II MHC antigens as well as other antigens on capillary endothelium including ICAM-1, LFA-3, and a novel antigen identified by E1.5. Expression of HLA-DR by peritubular capillaries was decreased during rejection, and this disappearance of peritubular capillaries with severe rejection was confirmed by loss of other markers of microvascular endothelium. These studies suggest peritubular capillaries may be the major target of the acute rejection response, and the techniques described allow assessment of degree of damage to these structures in renal allograft biopsies.
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Hemi-exon shuffling and site-directed mutagenesis have been used to determine which amino acid differences between HLA-A2.1 and HLA-A2.2 alter the CTL-defined epitopes on these two molecules. Two genes were constructed that encode novel molecules in which the effect of amino acid differences at residues 9, 43, and 95, or at residue 156 could be separately evaluated. Using both human and murine CTL that were specific for either HLA-A2.1 or HLA-A2.2, four types of epitopes were identified: 1) epitopes that were insensitive to substitutions at either residues 9, 43, and 95, or residue 156 but were lost when all four positions were changed; 2) epitopes that were dependent on the residues 9, 43, 95, but not residue 156; 3) epitopes that were dependent on residue 156, but not amino acid residues 9, 43, and 95; and 4) epitopes that were dependent on residues 9, 43, and 95, as well as amino acid residue 156. Overall, there was a roughly equal distribution of clones recognizing each of these types of epitopes. Additional molecules were constructed by hemi-exon shuffling between the HLA-A2.2 and HLA-A2.3 genes, and by site-directed mutagenesis, to analyze the epitopes recognized by two HLA-A2.2/A2.1 cross-reactive murine CTL that do not recognize HLA-A2.3. Although the epitopes recognized by these CTL were unaffected by changes occurring at residues 9, 43, and 95, or at residues 149, 152, and 156 alone, simultaneous changes in both of these regions acted in concert to destroy the epitopes. Both of the CTL recognized epitopes that were lost when substitutions were made at residues 9, 43, 95, 149, and 152. The epitope recognized by one of the CTL was also destroyed by the substitution of residues 9, 43, 95, 152, and 156. Overall, these results indicate that residues 9, 43, and 95, as well as residues in the alpha-helical region of the molecule, are all capable of contributing to the definition of the epitopes recognized by HLA-A2.1- and HLA-A2.2-specific CTL. They further indicate that some epitopes can be mapped to a particular region of the molecule, whereas other epitopes are formed through a complex interaction of residues in distant regions of the molecule.
Site-directed mutagenesis of HLA-A2.1 has been used to identify the amino acid substitutions in HLA-A2.3 that are responsible for the lack of recognition of the latter molecule by the HLA-A2/A28 specific antibody, CR11-351, and by HLA-A2.1 specific CTL. Three genes were constructed that encoded HLA-A2 derivatives containing one of the amino acids known to occur in HLA-A2.3: Thr for Ala149, Glu for Val152, and Trp for Leu156. Three additional genes were constructed that encoded the different possible combinations of two amino acid substitutions at these residues. Finally, a gene encoding all three substitutions and equivalent to HLA-A2.3 was constructed. These genes were transfected into the class I negative, human cell line Hmy2.C1R. Analysis of this panel of cells revealed that recognition by the antibody CR11-351 was completely lost when Thr was substituted for Ala149, whereas substitutions at amino acids 152 and 156, either singly or in combination, had no effect on the binding of this antibody. The epitopes recognized by the allogeneic and xenogeneic HLA-A2.1 specific CTL clones used in this study were all affected by either one or two amino acid substitutions. Of those epitopes sensitive to single amino acid changes, none were affected by the substitution of Thr for Ala149, whereas all of them were affected by at least one of the substitutions of Glu for Val 152 or Trp for Leu156. Overall, amino acid residue 152 exerted a stronger effect on the epitopes recognized by HLA-A2.1 specific CTL than did residue 156. Of those epitopes affected only by multiple amino acid substitutions, double substitutions at residues 149 and 152 or at 152 and 156 resulted in a loss of recognition, whereas a mutant with substitutions at residues 149 and 156 was recognized normally. This reemphasizes the importance of residue 152 and indicates that residue 149 can affect epitope formation in conjunction with another amino acid substitution. These results are discussed in the context of current models for the recognition of alloantigens and in light of the recently published three-dimensional structure of the HLA-A2.1 molecule.
We have used a cDNA library enriched for T cell-specific sequences to isolate genes expressed by T cells but not by other cell types. We report here one such gene, designated RANTES, which encodes a novel T cell-specific molecule. The RANTES gene product is predicted to be 10 kDa and, after cleavage of the signal peptide, approximately 8 kDa. Of the 68 residues, 4 are cysteines, and there are no sites for N-linked glycosylation. RANTES is expressed by cultured T cell lines that are Ag specific and growth factor dependent. RANTES expression is inducible in PBL by Ag or mitogen. In CTL, expression of RANTES decreases after stimulation with Ag and growth factors. Interestingly, RANTES was not expressed by any T cell tumor line tested. There is significant homology between the RANTES sequence and several other T cell genes, suggesting that they comprise a previously undescribed family of small T cell molecules.
The Daudi cell line is a B-lymphoblastoid line derived from a Burkitt lymphoma. Daudi cells lack cell surface expression of class I HLA molecules despite the presence of intracellular class I heavy chains. They have a defect in the gene encoding beta 2-microglobulin (beta 2m), resulting in lack of translatable mRNA for this protein. It has been thought that this deficiency is responsible for the lack of cell surface class I expression. However, data have recently been presented demonstrating that at least one mouse class I heavy chain can be expressed on the cell surface in the absence of beta 2m. These results raised the questions of whether the lack of beta 2m is the only defect in Daudi and whether transfer of this single gene could restore surface class I expression. We found that transfection of the mouse beta 2m gene into Daudi indeed rescued cell surface expression of class I HLA molecules, and that these molecules could be recognized both by monomorphic and allospecific mAbs. CTL clones specific for HLA-B17 or a determinant shared by HLA-B17 and HLA-A2 killed the Daudi cells transfected with the beta 2m gene, but not untransfected Daudi or Daudi transfected with vector alone. Mouse beta 2m on the transfected Daudi cells could exchange with human beta 2m when the cells were incubated in human serum. This exchange did not alter the ability of the cells to be killed by the specific CTLs. These results demonstrate that the lack of beta 2m is the sole reason for lack of surface class I molecules in Daudi cells, and that beta 2m is required for cell surface expression of the specific class I heavy chains of Daudi.
Because keratinocytes (KCs) express HLA-DR in a wide variety of skin diseases in which mononuclear leukocytes are observed in close apposition to KCs (i.e., graft-versus-host disease), and since gamma interferon (IFN-gamma) induces HLA-DR expression on KCs, we asked whether IFN-gamma treatment of KCs would influence the adherence of mononuclear leukocytes. When allogeneic peripheral blood mononuclear leukocytes (PBML) and a Leu-3+ T cell clone were coincubated with IFN-gamma-treated KCs (300 U/ml, 3 days), there was a marked increase in binding compared with nontreated KCs. Similar binding results were obtained using a cutaneous squamous carcinoma cell line (SCL-1) after IFN-gamma treatment. The IFN effect was relatively specific for IFN-gamma, as neither IFN-alpha nor -beta had any effect. Tumor necrosis factor exposure (500 U/ml, 3 days) increased the binding of the Leu-3+ T cell clone to both KCs and SCL-1 cells. Neutrophils displayed a less marked (but statistically significant) increase in binding to IFN-gamma-treated KCs. Using the Leu-3+ cell clone and SCL-1 cells, detailed kinetic analysis of the effect of IFN-gamma on binding was performed. The increased adherence between the cells began to appear after only 7 hours of treatment with r-IFN-gamma (300 U/ml) and reached a plateau at 48 hours, with significantly enhanced binding continuing for at least 48 hours after removal of IFN-gamma. The mechanism of binding was explored by preincubation of the PBML/Leu-3+ T cells with anti-LFA-1 (lymphocyte function-associated antigen) antibody (0.6-6.0 micrograms/ml), which totally inhibited the binding with no effect by anti-LFA-2 or -3 or class I or II antibodies despite documented binding of these antibodies to the cells. These results suggest that, after exposure to IFN-gamma, the ability of KCs to bind mononuclear leukocytes is strongly enhanced, and this adherence may be important in leukocyte trafficking in the skin as well as contributing to altered KC-leukocyte interaction, which may be of fundamental importance in a variety of skin disease.
During studies of T-cell recognition of autologous tumour cells, a number of tumour cell lines derived from patients with lymphoma proved to be poor stimulators of both autologous and allogeneic T-cell responses. Analysis of the tumour cell surface molecules indicated that expression of the lymphocyte-function-associated antigen, LFA-1, was lacking, whereas normal leucocytes from these patients expressed normal levels of LFA-1. Examination of other lymphoid tumours revealed that most high grade lymphomas, but not most low or intermediate grade lymphomas, do not express the LFA-1 molecule. Furthermore, in an initial survey, the tumours from 5 of 7 patients with non-relapsing large cell lymphomas expressed LFA-1 whereas only 3 of 18 patients with relapsing lymphomas had tumours that did so. These findings suggest that tumour cells lacking surface LFA-1 cannot initiate an effective immune response in vivo. This lack of immunogenicity might contribute to escape from immunosurveillance.
We have studied the interaction of HLA class I antigens with alloreactive cytotoxic T lymphocytes and monoclonal antibodies using site-directed mutagenesis and expression of an HLA-Aw68.1 gene. Two mutants containing distinct substitutions at polymorphic residues near the NH2-terminal end of the alpha 2 domain were made. One mutant with substitutions at positions 95 and 97 corresponding to residues found in HLA-A2.1 showed no alterations in binding of HLA-Aw68- or HLA-A2-specific monoclonal antibodies, but was reactive with some HLA-A2-specific CTL clones. A second mutant, in which glycine at position 107 was replaced with tryptophan found at that position in HLA-A2.1, was recognized by HLA-A2-specific CTL clones and HLA-A2, Aw69-specific monoclonal antibodies. Thus, substitution of a single amino acid residue at position 107 of the HLA-Aw68.1 molecule generates an allospecific determinant shared with HLA-A2.1 and recognized by both B and T lymphocytes.
The Leu-19 (NKH-1) antigen is expressed on human peripheral blood NK cells and a subset of peripheral blood cytotoxic T lymphocytes that kill "NK-sensitive" tumor cell targets without major histocompatibility complex restriction. In the present study, we demonstrate that the Leu-19 (NKH-1) antigen is also expressed on most interleukin 2 (IL 2) dependent T cell lines and clones that have been maintained in long term culture. The Leu-19 (NKH-1) antigen expressed on an antigen-specific, class I directed cytotoxic T lymphocyte cell line was an approximately 200,000 to 220,000 dalton protein, similar to Leu-19 (NKH-1) protein expressed on natural killer cells and KG1a, an immature stem cell leukemia cell line. Furthermore, Leu-19 (NKH-1) was expressed on both CD4+ and CD8+ IL 2 dependent T cell clones, and was present on both cytotoxic and non-cytotoxic T cell clones. Thus expression of Leu-19 (NKH-1) antigen on cultured cell lines does not directly correlate with cytotoxic function, antigenic specificity, or cell lineage.